mAb Penetration Research Articles

Exploring How Antibody Format Drives Clearance from the Brain.

Monoclonal antibodies (mAbs) have high binding specificity and affinity, making them attractive for treating brain diseases. However, their effectiveness is limited by poor blood-brain barrier (BBB) penetration and rapid central nervous system (CNS) clearance. Our group identified blood-brain barrier modulator (BBBM) peptides that improved mAb penetration across the BBB into the brain. In this study, we investigated the pharmacokinetics of a mAb delivered to the brain using BBBMs after intravenous (IV) administration and explored the impact of antibody format (size, neonatal Fc receptor (FcRn) binding, hyaluronic acid binding) on brain clearance following direct injection into the central nervous system (CNS) via intracerebroventricular (ICV) injection. IRDye800CW-labeled antibodies were administered into C57BL/6 mice via ICV or IV injection, and organ concentrations were measured after various time points. When a mAb was coadministered with a BBBM peptide, the permeation of mAb across the BBB was increased compared to mAb alone at early time points; however, the mAb was cleared within 2 h from the brain. ICV experiments revealed that an antibody Fab fragment had a higher brain exposure than a mAb, and that a Fab fused to a hyaluronic acid binding domain (Fab-VG1) showed remarkable improvement in brain exposure. These findings suggest that BBBMs and antibody format optimization may be promising strategies for enhancing brain retention of therapeutic antibodies.

Modeling and Molecular Dynamic Simulation of F(ab')2 Fragment of Nimotuzumab for Lung Cancer Diagnostics.

Lung cancer is one of the leading causes of cancer-related deaths in the world among both men and women. Several studies in the literature report that overexpression and mutation of the epidermal growth factor receptor (EGFR) are implicated in the pathogenesis of some lung cancers. Nimotuzumab is a humanized monoclonal antibody (mAb) that inhibits EGF binding because it binds to the extracellular domain of the EGFR. Nimotuzumab requires bivalent binding for stable attachment to cellular surface, which leads to nimotuzumab selectively binding to cells that express mAbs of moderate to high EGFR levels, and this could explain its low toxicity. This property has an advantage for development of nimotuzumab as a therapeutic and diagnostic agent. Monoclonal antibodies are large in size (150 kDa), thus penetrating slowly and residing in the blood for extended periods of time (from days to weeks); their use in imaging studies can result in low signal-to-background ratios and poor image quality. A reduction in the size of the immunoglobulin molecule has also been proposed as a means for increasing tumor penetration by mAbs. Nevertheless, it is known that the penetration of mAb into tumor cell is slow, due to its high molecular weight. Therefore, mAb is not very attractive to be used for imaging diagnostic purpose because of its kinetics and potential to elicit antibody response. The objective of this research was to study the homology modeling of a simpler functional molecule based on nimotuzumab, which consists of 2 antigen-binding fragments (Fab), namely, F(ab′)2, using MODELER. The crystal structure of Fab of nimotuzumab from protein data bank was used as a template to construct the model of F(ab′)2. Molecular dynamic simulation was performed to evaluate the stability of F(ab′)2 and conformational changes of F(ab′)2 in simulation. The result showed the dynamic behavior of antigen-binding site region of F(ab′)2 throughout simulation. This result is expected to be useful in the further development of F(ab′)2 fragment nimotuzumab as a lung cancer diagnostic.

Impact of blood-brain barrier permeabilization induced by ultrasound associated to microbubbles on the brain delivery and kinetics of cetuximab: An immunoPET study using 89Zr-cetuximab

Epidermal growth factor receptor (EGFR), involved in cell proliferation and migration, is overexpressed in ~50% of glioblastomas. Anti-EGFR based strategies using monoclonal antibodies (mAb) such as cetuximab (CTX) have been proposed for central nervous system (CNS) cancer therapy. However, the blood-brain barrier (BBB) drastically restricts their brain penetration which limits their efficacy for the treatment of glioblastomas. Herein, a longitudinal PET imaging study was performed to assess the relevance and the impact of focused ultrasound (FUS)-mediated BBB permeabilization on the brain exposure to the anti-EGFR mAb CTX over time. For this purpose, FUS permeabilization process with microbubbles was applied on intact BBB mouse brain before the injection of 89Zr-labeled CTX for longitudinal imaging monitoring. FUS induced a dramatic increase in mAb penetration to the brain, 2 times higher compared to the intact BBB. The transfer of 89Zr-CTX from blood to the brain was rendered significant by FUS (kuptake = 1.3 ± 0.23 min−1 with FUS versus kuptake = 0 ± 0.006 min−1 without FUS). FUS allowed significant and prolonged exposure to mAb in the brain parenchyma. This study confirms the potential of FUS as a target delivery method for mAb in CNS.

Impact of global ultrasound-induced blood-brain barrier opening on the distribution of full monoclonal antibody cetuximab

Aim/Introduction Epidermal growth factor receptor (EGFR), involved in cell proliferation and migration, is overexpressed in 50% of glioblastomas that amplify wild type EGFR. This suggests a relevance for molecularly-targeted therapy using anti-EGFR based strategies using monoclonal antibodies (mAb), such as cetuximab, for therapy. Clinical trials have shown the beneficial effects of cetuximab in various EGFR-expressing peripheral tumors, but failed in patients with recurrent glioblastoma. One of the main issues is the poor penetration of mAb into the central nervous system (CNS) due to the presence of the blood-brain barrier (BBB). Recently, ultrasound (US) in combination with microbubbles has been used to transiently open the BBB and promote the delivery of therapeutic agent to CNS. Non-invasive, dynamic and quantitative methods such as PET imaging using radiolabeled mAb may provide a translational imaging method to address the efficacy of US for the delivery of mAb into brain. Materials and Methods Cetuximab was conjugated to DFO and labeled with 89Zr at 0.5 MBq/μg. Affinity comparison between human and murine EGFR with cetuximab has been done to accurately measure kinetic constants. In vivo PET imaging and biodistribution were conducted in healthy nude male mice (no US vs US groups, n = 8) in order to follow the kinetics of the 89Zr-Cetuximab overtime. Brain uptake of 89Zr-Cetuximab was initially monitored by whole body dynamic PET, followed by static PET-CT imaging at different time points post-injection. The TACs allowed the determination of the transfer rate constants between the blood and the brain. Results Cetuximab exhibits an approximate 10-fold greater affinity with human EGFR compared to mouse counterpart. In control mice, the brain distribution of 89Zr-Cetuximab was negligible (transfer rate = 0 ± 0.006 min-1). The transfer rate increased up to 1.3 ± 0.23 min-1 when US were applied. The brain uptake without US remained constant at 0.7 ± 0.09%ID/cc overtime whereas it reached 1.34 ± 0.15%ID/cc at 1 h and stay stable during 48 hours with US (1.36% ± 0.16%ID/cc). After 72 h post-US, no significant difference between the US and the control cohorts was observed. Conclusion Here, PET imaging was successfully combined with therapeutic US to demonstrate the efficiency of this strategy for mAb delivery into brain which raises potential application for tumor treatment.

Abstract LB-219: Derivation of mathematical modeling for the diffusion and penetration of monoclonal antibody delivery into tumor

Abstract Background: Commonly malignant solid tumor have an abundant tumor-associated cellular matrix (ECM). Although radioimmunothrepy (RIT) have proven to be effective and precise treatment in cancer, yet RIT has a limited performance in solid tumor, due to highly distributed ECM inside solid tumor tissue. Previously, we designed the combination RIT to enhance the penetration of monoclonal antibody (mAb), where we investigated that, using Relaxin which contain oncolytic adenovirus (OA) as combination therapy decreases tumor associated ECM. In this study, we developed mathematical modeling to explain and describe the diffusion and penetration of mAb inside the tumor with ECM. To obtain the model parameters in our developed mathematical model, we performed related experiments such as immunofluorescence and qRT-PCR. Methods: We developed the reaction-diffusion partial differential equations (PDE) for describing the dependency of cancer cell, ECM density, diffusion and penetration of mAb and ECM modulated therapy that include our experimental data, also to overcome the classical limitation of PDE method. Model included the rapid decrease of ECM by OA, and density dependent diffusion coefficient. Initial condition for solving the PDE was derived using our experimental result. Diffusion coefficient for mAb and OA was modelled for simulating actual behavior of mAb and OA. Various condition of ECM density, and structure were modelled that derives the actual result of distribution of collagen. To confirm the mathematical prediction, we designed in vitro experiment with same condition as used as “in model”. Results: Using our developed model, we successfully described relationship between ECM and mAb transport by OA, and investigated the change of ECM. Diffusion and penetration of mAb and OA were well described from tumor vessel and tumor surface. Regardless of ECM structure, all ECM structure were degraded after spreading of OA. In addition, our model simulation results predicted that mAb uptake dramatically increased in combination approach and was more than mAb alone treatment. Our developed mathematical model predicted the optimal schedule of OA pretreatment and it shows great results more than mAb pretreatment. Pretreatment results with OA shows a great anti-tumor effect more than mAb pretreatment in experimental data. Our model could well predicted and matched the experimental results. Collectively, in our study, we suggested new theoretical mathematical model framework to explain various ECM condition with drug transport Citation Format: Hyeongi Kim, Yangjin Kim, Yong-Jin Lee, Jin-su Kim. Derivation of mathematical modeling for the diffusion and penetration of monoclonal antibody delivery into tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-219.

The Alpha Emitter Astatine-211 Targeted to CD38 Can Eradicate Multiple Myeloma in Minimal Residual Disease Models

IntroductionMultiple myeloma (MM) is considered incurable but patients achieving minimal-residual disease (MRD) negative status following treatment have significantly better overall and progression-free survival. MM is highly heterogeneous both between and within patients, limiting the curative potential of novel agents targeting specific pathways. However all MM is highly sensitive to radiation. The α-emitter astatine-211 (211At) deposits a very large amount of energy (~100 keV/μm) within a few cell diameters (50-90 μm) resulting in irreparable double strand DNA breaks, making 211At, targeted to MM cells, particularly suited to eliminating MRD. CD38 is expressed on malignant plasma cells regardless of mutational status, and CD38 monoclonal antibodies (mAbs) constitute a proven targeted therapy for MM but do not alone eradicate disease. We proposed that 211At conjugated to an anti-CD38 mAb could effectively eliminate MM MRD, and tested this hypothesis in cellular and murine models.MethodsWe conjugated the anti-CD38 mAb OKT10 and an isotype matched control mAb, BHV1, to the amine-reactive labeling agent B10-NCS and labeled the final constructs with 211At. To assess in vitro cell binding we incubated each labeled construct with CD38+ cell lines, washed, and then measured cell pellet radioactivity in a gamma counter. To assess cytotoxicity we incubated CD38+ and CD38- cell lines with unlabeled or 211At-labeled OKT10-B10 for 60 hrs, then assayed viability. NOD.Cg-Rag1tm1MomIl2rgtm1Wjl/SzJ (NRG) mice bearing H929luc or OPM-2luc MM xenografts were generated by subcutaneous (SQ) flank injection of 107 cells 7 days prior to treatment. MRD was modeled by intravenous (IV) injection of 2.5 - 5 x 105 cells 5 days prior to treatment. Radioimmunotherapy (RIT) was administered by IV injection of 7.5 - 45 µCi of 211At-OKT10-B10 or 211At-BHV1-B10. For biodistribution studies (n = 5/group) mouse tissues were harvested 24 hrs post RIT and measured in a gamma counter. For therapy studies (n = 8-10/group), all mice received syngeneic bone marrow transplant 3 days post RIT. Disease progression was assessed by tumor dimensions, luminescence imaging and survival.Results211At-CD38 mAb selectively bound and killed CD38+ but not CD38- MM cells in vitro. In vivo, biodistribution experiments demonstrated that 211At-CD38 RIT delivered 2.4 times more radiation to MM xenografts than did control 211At-BHV1 RIT (p = 0.007), and delivered significantly higher dose to tumor than to healthy tissues including lung (p = 0.04) and kidney (p = 0.015). In murine therapy studies, 211At-CD38 RIT at 15 - 45 µCi at least doubled median survival relative to untreated controls in each of two MM SQ xenograft models (p < 0.003). However, no mice in these models achieved complete remission and all eventually died of disease. In contrast, therapy studies using MRD models showed that 30 µCi 211At-CD38 RIT eliminated detectable disease in 80% of mice at day 21, compared to 20% of mice receiving nontargeted 211At RIT and 0% of untreated mice. At day 130, 50% of the 30 µCi 211At-CD38-RIT mice remained alive and disease-free, while all nontargeted and untreated mice died before day 85 (Fig. 1, survival of 30 µCi > 15 µCi 211At-CD38-RIT [p = 0.016] and all other groups [p < 0.0007]). The impact of therapy on body weight as well as hematopoietic, liver and kidney function was mild and returned to normal within 32 days of treatment.ConclusionsThe efficacy of CD38 targeted 211At appears to be a function of disease distribution and malignant plasma cell access, as compellingly demonstrated by our models. Bulky tumor geometry reduces mAb penetration. In contrast, the isolated cells and small tumor clusters that define MRD are readily accessible to mAbs, creating optimal conditions for α-emitter cell kill. In an era of highly potent MM therapy, preventing relapse remains frustratingly rare. Our approach is both agnostic to high-risk cytogenetic features and offers the potential to eliminate all residual MM cell clones. These encouraging findings will be explored in a clinical trial of 211At-CD38 RIT. [Display omitted] DisclosuresOrozco:Actinium Pharmaceuticals: Research Funding. Jones:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Till:Mustang Bio: Patents & Royalties, Research Funding. Gopal:Teva: Research Funding; Spectrum: Research Funding; Janssen: Consultancy, Research Funding; BMS: Research Funding; Incyte: Consultancy; Gilead: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Brim: Consultancy; Pfizer: Research Funding; Aptevo: Consultancy; Takeda: Research Funding; Merck: Research Funding; Asana: Consultancy. Green:Juno Therapeutics: Patents & Royalties, Research Funding.

Systemic infusions of anti-interleukin-1β neutralizing antibodies reduce short-term brain injury after cerebral ischemia in the ovine fetus

Perinatal hypoxic-ischemic reperfusion (I/R)-related brain injury is a leading cause of neurologic morbidity and life-long disability in children. Infants exposed to I/R brain injury develop long-term cognitive and behavioral deficits, placing a large burden on parents and society. Therapeutic strategies are currently not available for infants with I/R brain damage, except for hypothermia, which can only be used in full term infants with hypoxic-ischemic encephalopathy (HIE). Moreover, hypothermia is only partially protective. Pro-inflammatory cytokines are key contributors to the pathogenesis of perinatal I/R brain injury. Interleukin-1β (IL-1β) is a critical pro-inflammatory cytokine, which has been shown to predict the severity of HIE in infants. We have previously shown that systemic infusions of mouse anti-ovine IL-1β monoclonal antibody (mAb) into fetal sheep resulted in anti-IL-1β mAb penetration into brain, reduced I/R-related increases in IL-1β expression and blood-brain barrier (BBB) dysfunction in fetal brain. The purpose of the current study was to examine the effects of systemic infusions of anti-IL-1β mAb on short-term I/R-related parenchymal brain injury in the fetus by examining: 1) histopathological changes, 2) apoptosis and caspase-3 activity, 3) neuronal degeneration 4) reactive gliosis and 5) myelin basic protein (MBP) immunohistochemical staining. The study groups included non-ischemic controls, placebo-treated ischemic, and anti-IL-1β mAb treated ischemic fetal sheep at 127days of gestation. The systemic intravenous infusions of anti-IL-1β mAb were administered at fifteen minutes and four hours after in utero brain ischemia. The duration of each infusion was two hours. Parenchymal brain injury was evaluated by determining pathological injury scores, ApopTag® positive cells/mm2, caspase-3 activity, Fluoro-Jade B positive cells/mm2, glial fibrillary acidic protein (GFAP) and MBP staining in the brains of fetal sheep 24h after 30min of ischemia. Treatment with anti-IL-1β mAb reduced (P<0.05) the global pathological injury scores, number of apoptotic positive cells/mm2, and caspase-3 activity after ischemia in fetal sheep. The regional pathological scores and Fluoro-Jade B positive cells/mm2 did not differ between the placebo- and anti-IL-1β mAb treated ischemic fetal sheep. The percent of the cortical area stained for GFAP was lower (P<0.05) in the placebo ischemic treated than in the non-ischemic group, but did not differ between the placebo- and anti-IL-1β mAb treated ischemic groups. MBP immunohistochemical expression did not differ among the groups. In conclusion, infusions of anti-IL-1β mAb attenuate short-term I/R-related histopathological tissue injury, apoptosis, and reduce I/R-related increases in caspase-3 activity in ovine fetal brain. Therefore, systemic infusions of anti-IL-1β mAb attenuate short-term I/R-related parenchymal brain injury in the fetus.

Abstract 2663: Hyperthermia improves Cetuximab accumulation in pancreatic cancer mouse model

Abstract Objective: Histopathological characteristics of pancreatic carcinoma are the presence of abundant stroma that prevent mAb diffusion and consequently hampers mAb from direct attacking cancer cells. With a strong conviction that additional tool that improve mAb penetration and diffusion in dense fibroblastic solid cancer should indispensable for better therapeutic effect, we focused and employed the simple but strongest modalities, heat. Heat might increase tumor blood flow and tumor vessel permeability, leading enhanced accumulation of anticancer mAb. In this study, we aimed the increased accumulation of mAb in mouse pancreatic subcutaneous models and also boosted antitumor effects by applying moderate heat. Methods: Heat generation was completed by hot bathtub method for subcutaneous tumor in nude mice leg. Three human pancreatic cell of different stromal amount were employed (abundant: Capan-1, moderate: BxPC-3, and scant: MIAPaCa-2). At the timing of each tumors growth up to 70mm3, Cetuximab (1 mg/kg) was systematically administrated via caudal vein, subsequently applying heat to tumors for 30 min at three different temperatures of 25°C (control temperature), 37°C (intraabdominal organs), or 41°C (hyperthermia) (n = 5 each). The amount of accumulated Cetuximab were quantified by the fluorescent intensity of Alexa 488 against anti-human IgG at 24 hr, followed by figuring up the intensity of one cancer cell by dividing the total intensity value by the number of a cancer cell nuclei stained with DAPI. Enhanced antitumor effects, among irradiating various heat doses, were evaluated by the tumor volume over time. Results: In Capan-1, the fluorescent intensity per a cancer cell at 25°C, 37°C, 41°C were 675, 1130, 2332 respectively. Similarly, those in BxPC-3 were 673, 1347, 1573, and in MIAPaCa-2 were 1632, 1921, 1949, respectively. In Capan-1 and BxPC-3, the mean tumor growth on day 40 were significantly inhibited at 37°C group (934, 189 mm3), and 41°C group (491, 121 mm3) in compared with 25°C group (1385, 470 mm3) respectively. In MIAPaCa-2, however, that on day 50 was not inhibited at 37 degrees group (109 mm3), and 41 group (151 mm3) as compared with 25 group (123 mm3). Discussion: We demonstrated that hyperthermia actually contributed to measurable add-on antitumor effect for two among three pancreatic cancer models. Intending to provide more add-on effect, we are now evaluating the combined effects of hyperthermia plus applying a cyclic tumor-penetrating peptide (iRGD), which improved anticancer mAb penetration to cancer cells by binding to integrin αv/β3 or β5 and to neuropillin-1 on cancer cells. We suggested that this novel combined strategy would remarkably contribute to the enhanced accumulation of anticancer mAb in tumors and to the enhanced antitumor effects. Citation Format: Ryoichi Miyamoto, Tatsuya Oda, Shinji Hashimoto, Yoshimasa Akashi, Tomohiro Kurokawa, Yuki Inagaki, Nobuhiro Ohkohchi. Hyperthermia improves Cetuximab accumulation in pancreatic cancer mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2663. doi:10.1158/1538-7445.AM2014-2663

Indirect targeting of IGF receptor signalingin vivoby substrate-selective inhibition of PAPP-A proteolytic activity

The insulin-like growth factor (IGF) signaling pathway is involved in certain human cancers, and the feasibility of directly targeting the IGF receptor has been actively investigated. However, recent evidence from clinical trials suggests that this approach can be problematic. We have developed an alternative strategy to indirectly inhibit the IGF signaling by targeting the metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). PAPP-A associated with the cell surface cleaves IGF binding protein-4 (IGFBP-4), when IGF is bound to IGFBP-4, and thereby increases IGF bioavailability for receptor activation in an autocrine/paracrine manner. We hypothesized that inhibition of PAPP-A would suppress excessive local IGF signaling in tissues where this is caused by increased PAPP-A proteolytic activity. To test this hypothesis, we developed an inhibitory monoclonal antibody, mAb 1/41, which targets a unique substrate-binding exosite of PAPP-A. This inhibitor selectively and specifically inhibits proteolytic cleavage of IGFBP-4 with an inhibitory constant (Ki) of 135 pM. In addition, it inhibited intracellular signaling of the IGF receptor (AKT phosphorylation) in monolayers of A549 cells, an IGF-responsive lung cancer-derived cell line found to express high levels of PAPP-A. We further showed that mAb 1/41 is effective towards PAPP-A bound to cell surfaces, and that it is capable of inhibiting PAPP-A activity in vivo. Using a murine xenograft model of A549 cells, we demonstrated that mAb 1/41 administered intraperitoneally significantly inhibited tumor growth. Analysis of xenograft tumor tissue recovered from treated mice showed penetration of mAb 1/41, reduced IGFBP-4 proteolysis, and reduced AKT phosphorylation. Our study provides proof of concept that IGF signaling can be selectively reduced by targeting a regulatory proteinase that functions extracellularly, upstream of the IGF receptor. PAPP-A targeting thus represents an alternative therapeutic strategy for inhibiting IGF receptor signaling.

Influence of Affinity and Antigen Internalization on the Uptake and Penetration of Anti-HER2 Antibodies in Solid Tumors

Antibody drugs are widely used in cancer therapy, but conditions to maximize tumor penetration and efficacy have yet to be fully elucidated. In this study, we investigated the impact of antibody binding affinity on tumor targeting and penetration with affinity variants that recognize the same epitope. Specifically, we compared four derivatives of the C6.5 monoclonal antibody (mAb), which recognizes the same HER2 epitope (monovalent K(D) values ranging from 270 to 0.56 nmol/L). Moderate affinity was associated with the highest tumor accumulation at 24 and 120 hours after intravenous injection, whereas high affinity was found to produce the lowest tumor accumulation. Highest affinity mAbs were confined to the perivascular space of tumors with an average penetration of 20.4 ± 7.5 μm from tumor blood vessels. Conversely, lowest affinity mAbs exhibited a broader distribution pattern with an average penetration of 84.8 ± 12.8 μm. In vitro internalization assays revealed that antibody internalization and catabolism generally increased with affinity, plateauing once the rate of HER2 internalization exceeded the rate of antibody dissociation. Effects of internalization and catabolism on tumor targeting were further examined using antibodies of moderate (C6.5) or high-affinity (trastuzumab), labeled with residualizing ((111)In-labeled) or nonresidualizing ((125)I-labeled) radioisotopes. Significant amounts of antibody of both affinities were degraded by tumors in vivo. Furthermore, moderate- to high-affinity mAbs targeting the same HER2 epitope with monovalent affinity above 23 nmol/L had equal tumor accumulation of residualizing radiolabel over 120 hours. Results indicated equal tumor exposure, suggesting that mAb penetration and retention in tumors reflected affinity-based differences in tumor catabolism. Together, these results suggest that high-density, rapidly internalizing antigens subject high-affinity antibodies to greater internalization and degradation, thereby limiting their penetration of tumors. In contrast, lower-affinity antibodies penetrate tumors more effectively when rates of antibody-antigen dissociation are higher than those of antigen internalization. Together, our findings offer insights into how to optimize the ability of therapeutic antibodies to penetrate tumors.

Computational model predicts effective delivery of 188-Re-labeled melanin-binding antibody to metastatic melanoma tumors with wide range of melanin concentrations

Metastatic melanoma is almost always deadly and new methods of treatment are urgently needed. Recently, we established the feasibility of radioimmunotherapy (RIT) for experimental melanoma in mice using a 188-rhenium (188Re)-labeled monoclonal antibody (mAb) 6D2 (IgM) to melanin. Our objective was to determine the effects of varying tumor melanin concentration and of different diffusivities and lymphatic clearance rates of the normal tissue, on the absorbed dose to the tumor in simulated therapy, in preparation for a clinical trial of RIT for melanoma. Using finite element analysis (FEA), we created a pharmacokinetic model that describes melanin-targeting RIT of a melanoma micrometastasis (1.3-mm radius) imbedded in normal tissue (14.3-mm radius). Our method incorporates antibody plasma kinetics, transcapillary transport, interstitial diffusion, and lymphatic clearance. Michaelis-Menten kinetics was used to model mAb binding to tumor melanin for melanin concentrations of 76, 7.6, 0.76, 0.076, and 0.0076 micromol/l. An absorbed dose was calculated, after accounting for direct and crossfire irradiation, on the basis of a 7.4-GBq intravenous dose of 188Re-6D2. The results showed that penetration of mAb into the tumor was inversely proportional to tumor melanin concentration. Decreased diffusivity and increased lymphatic clearance of the surrounding normal tissue decreased the dose to the tumor. The formation of mAb-melanin complex was remarkably similar within a 1000-fold range of melanin concentration, resulting in total doses of 2840, 2820, 2710, and 1990 cGy being delivered to tumors with melanin concentrations of 76, 7.6, 0.76, and 0.076 micromol/l, respectively. In conclusion, RIT of metastatic melanoma can be effective over a wide range of tumor melanin concentrations. The results can be useful in the design of a clinical trial of melanin-targeting RIT in patients with metastatic melanoma.

Destructive effects of murine arthritogenic antibodies to type II collagen on cartilage explants in vitro

Certain monoclonal antibodies (mAbs) to type II collagen (CII) induce arthritis in vivo after passive transfer and have adverse effects on chondrocyte cultures and inhibit self assembly of collagen fibrils in vitro. We have examined whether such mAbs have detrimental effects on pre-existing cartilage. Bovine cartilage explants were cultured over 21 days in the presence of two arthritogenic mAbs to CII (CIIC1 or M2139), a non-arthritogenic mAb to CII (CIIF4) or a control mAb (GAD6). Penetration of cartilage by mAb was determined by immunofluorescence on frozen sections and correlated with changes to the extracellular matrix and chondrocytes by morphometric analysis of sections stained with toluidine blue. The effects of mAbs on matrix components were examined by Fourier transform infrared microspectroscopy (FTIRM). A possible role of Fc-binding was investigated using F(ab)2 from CIIC1. All three mAbs to CII penetrated the cartilage explants and CIIC1 and M2139, but not CIIF4, had adverse effects that included proteoglycan loss correlating with mAb penetration, the later development in cultures of an abnormal superficial cellular layer, and an increased proportion of empty chondrons. FTIRM showed depletion and denaturation of CII at the explant surface in the presence of CIIC1 or M2139, which paralleled proteoglycan loss. The effects of F(ab)2 were greater than those of intact CIIC1. Our results indicate that mAbs to CII can adversely affect preformed cartilage, and that the specific epitope on CII recognised by the mAb determines both arthritogenicity in vivo and adverse effects in vitro. We conclude that antibodies to CII can have pathogenic effects that are independent of inflammatory mediators or Fc-binding.

The effect of antibody protein dose on the uniformity of tumor distribution of radioantibodies: an autoradiographic study.

The inaccessibility of radiolabeled antibody to poorly vascularized regions of solid tumors may reduce the therapeutic efficacy of these macromolecules. Theoretical mathematical models have predicted that increasing the protein dose administered would reduce the heterogeneity of radioantibody distribution. This investigation was undertaken to evaluate this hypothesis in experimental animal models. We have utilized the technique of macroautoradiography to demonstrate an increase in tumor penetration of the lower-affinity 125I-labeled NP-4 or higher-affinity Immu-14 anti-carcinoembryonic antigen (anti-CEA) mAbs into small (60.25-0.4 g) and large (0.8-1.5 g) GW-39 and LS174T human colonic xenografts, grown subcutaneously in the nude mouse, when 400 micrograms unlabeled antibody is administered simultaneously with 10 micrograms (100 microCi) radioantibody. Further increases in protein to 800 micrograms result in a reduction in total tumor uptake of the antibody. These in a reduction in total tumor uptake of the antibody. These differences in mAb distribution could be visualized as early as 1 day after antibody injection. Improved mAb penetration was also achieved for the Mu-9 anti-CSAp (anti-mucin) antibody using 800 micrograms unlabeled antibody. An irrelevant antibody (AFP-7-31) was found to be homogeneously distributed 3 days after injection, even at a low protein dose. Attempts to improve mAb penetration by increasing the protein dose in the GS-2 colorectal tumor, a model that has low NP-4 accretion as a result physiological barriers separating antibody from antigen, were not successful. These results suggest that a more homogeneous distribution of radioantibody can be achieved by carefully selecting a dose of unlabeled antibody to coadminister. Work is currently in progress to determine the effect of improved tumor distribution of radioantibody on the therapeutic potential of a single dose of radioantibody.